Specific release systems in the colon have been proven to have significant theracanic advantages.
A large number of colonic illnesses could effectively be treated more efficaciously if the active ingredient were released locally. This is the case, inter alia, for Crohn's disease, ulcerative colitis, colorectal cancer and constipation.
Colon-targeted release could also be interesting when, from the theracanic point of view, a delay in absorption is necessary, in particular in the treatment of pathologies such as nocturnal asthma or angor (Kinget R. et al. (1998), Colonic Drug Targeting, Journal of Drug Targeting, 6,129).
The administration of polypeptidic active ingredients occurs essentially parenterally, which is painful and the origin of poor observation of treatment. For some years now there has been an interest in using the colon as an absorption site for peptidic active ingredients (analgesic, contraceptive, vaccine, insulin . . . ). The absorption of peptides in the colon seems effectively better than at other sites of the digestive tract, in particular due to proteolytic activity clearly weaker than in the small intestine and in the absence of peptidasic activity associated with the membrane of the epithelial colonic cells.
During oral administration of antibiotics, these pass through the stomach and are then absorbed in the small intestine to diffuse in the entire organism and treat the infectious focus for which they have been administered. All the same, a fraction of ingested antibiotics (whereof the importance varies with the characteristics peculiar to each type of antibiotic) is not absorbed and continues its progress to the colon before being eliminated in the stool. These residual antibiotics are reunited, in the small intestine, by a fraction of the antibiotics absorbed, but which are re-excreted in the digestive tract by way of biliary elimination. This fraction is of variable importance as a function of metabolism and elimination paths of each antibiotic. Finally, for certain antibiotics, a fraction of the dose absorbed is eliminated directly by the intestinal mucous in the lumen of the digestive tract. Since the antibiotics had been administered orally or parenterally, an active residual fraction is generally found in the colon. This holds true, to varying degrees, for the vast majority of the families of antibiotics utilised in therapeutics, the only notable exception being the family of amino-glycosides for which intestinal excretion is negligible. For the other antibiotics, intestinal excretion of a residual antibiotic activity will have different consequences, all harmful. In effect, in the colon there is a complex (several hundreds of different bacterial species) and very dense (more than 1011 bacteria per gram of colonic content) bacterial ecosystem which will be affected by the arrival of active residues of antibiotics. The following is observed:
1) imbalance of flora which would be the main cause of banal diarrhoea sometimes following ingestion of antibiotics (Bartlett J. G. (2002) Clinical practice. Antibiotic associated diarrhea, New England Journal of Medicine, 346, 334). Even though this diarrhoea is generally not serious and quickly abates, either spontaneously, or when treatment is discontinued, it is nevertheless badly received by patients and adds to the discomfort of the basic illness for which antibiotics have been prescribed;
2) perturbation of the functions of resistance to colonisation by exogenic bacteria (or “barrier effect”) with the possibility of increased risk of infection, for example, alimentary intoxication to Salmonella (Holmberg S. D. et al. (1984) Drug resistant Salmonella from animals fed antimicrobials, New England Journal of Medicine, 311, 617);
3) selection of micro-organisms resistant to antibiotics. The latter can be of various types:
a) they can first be pathogenic bacteria, such as for example Clostridium difficile, a species capable of secreting toxins causing redoubtable colitis known as pseudomembranous (Bartlett J. G. (1997) Clostridium difficile infection: pathophysiology and diagnosis, Seminar in Gastrointestinal Disease, 8,12);
b) they can also be relatively slightly pathogenic micro-organisms but whereof the multiplication can lead to surrounding infection (vaginal Candidosis or Escherichia coli resistant cystitis).
c) they can finally be non-pathogenic commensal resistant bacteria but whereof the multiplication and faecal elimination will boost dissemination in the environment. As it is, these resistant commensal bacteria can constitute an important source of resistance mechanisms for pathogenic species. This risk is currently considered to be major in terms of the worrying character in the evolution to multiresistance of numerous pathogenic species for humans.
Numerous strategies exploiting the diverse physiological parameters of the digestive tract have therefore been envisaged with a view to releasing active ingredients in the colon. Studies have in particular been carried out by means of administration systems based on (1) utilisation of polymers sensitive to variations in pH, (2) release forms dependent on time, (3) prodrugs or again polymers degradable by bacteria in the flora.
(1) Systems Based on Variations in pH.
The pH in the stomach is of the order of 1 to 3 but it increases in the small intestine and colon to reach values close to 7 (Hovgaard L. et al. (1996) Current Applications of Polysaccharides in Colon Targeting, Critical Reviews in Theracanic Drug Carrier Systems, 13,185). For an active ingredient to reach the colon, without undergoing these variations in pH, it is possible to administer it in the form of tablets, gels or spheroids coated in a pH-dependent polymer, insoluble in acid pH but soluble in neutral or alkaline pH (Kinget et al. op cit). The most commonly used polymers are derivatives of methacrylic acid, Eudragite L and S (Ashford M. et al. (1993), An in vivo investigation into the suitability of pH-dependent polymers for colonic targeting, International Journal of Pharmaceutics, 95,193 and 95,241; and David A. et al. (1997) Acrylic polymers for colon-specific drug delivery, S.T.P. Pharma Sciences, 7, 546).
Given the important inter- and intra-individual variability of the values of pH at the gastro-intestinal tract level, pH-dependent polymers do not represent the best means for obtaining specific release in the colon (Ashford M. et al., op cit.).
(2) Systems Based on Transit Time.
The formulation of these systems is such that it allows release of the active ingredients after a predefined lag time. So as to release the active ingredient in the colon, these forms must still resist the acid environment of the stomach and enter a silent phase of a predetermined time, before releasing the active ingredient, corresponding to the transit time from the mouth to the terminal ileon (Gazzaniga A. et al. (1995) Time-dependent oral delivery systems for colon targeting, S.T.P. Pharma Sciences, 5,83 and 108, 77; Liu P. et al. (1999) Alginate/Pectin/Poly-L-lysine particulate as a potential controlled release formulation, J. Pharm. Pharmacol., 51, 141; Pozzi F. et al. (1994) The Time Clock system: a new oral dosage form for fast and complete release of drug after predetermined lag time, Journal of Controlled Release, 31,99).
Pulsincap® by Scherer was one of the first formulations of this type (international patent application WO90/09168). It has the appearance of a gel whereof the body is insoluble in water. The active ingredient is maintained in the body by a hydrogel stopper placed in the head of the hydrosoluble gel. The whole is coated in a gastro-resistant film. After dissolution of the head in the small intestine, the stopper swells on contact with digestive juices. When the latter reaches a critical swelling threshold it is ejected, thus allowing release of the active ingredient. The ejection time is controlled by the properties of the hydrogel constituting the stopper.
Systems based on transit time nevertheless offer numerous disadvantages (variations in time for emptying of stomach and transit time, retention phenomena in the ileo-caecal valve (Kinget R., op. cit.), causing a lack in specificity and preventing validation of the latter as specific release systems in the colon. Finally, large-scale production of this type of system is difficult to envisage, as this would require and costly significant adaptation of industrial technologies.
Recently a novel form for colonic targeting has been developed, “Colon-targeted Delivery Capsule” (CTDC) (Ishibashi T. et al. (1998) Design and evaluation of a new capsule-type dosage form for colon-targeted delivery of drugs, International Journal of Pharmaceutics, 168,31 and 57,45). The CTDC is a system bringing together the pH-dependent factor and the time-dependent factor. It is in the form of a classic gel encasing the active ingredient and an organic acid (succinic acid), covered in 3 layers.
(3) Systems Based on Enzymatic Activity of the Microbial Colonic Flora.
3.1. Prodrugs.
Prodrugs have largely been studied for colonic targeting of various active ingredients (anti-inflammatory non-steroidal and steroidal, spasmolytic, . . . ). These systems are based on the capacity of the enzymes produced by the colonic flora for degrading prodrugs in order to release the active form of the active ingredient.
Numerous prodrugs based on the action of the bacterial azoreductases in particular have been developed with the aim of releasing in the colon active ingredients such as 5-aminosalicylic acid (5-ASA) utilised in the treatment of local pathologies such as Crohn's disease or ulcerative colitis (Peppercorn M. A. et al. (1972) The role of intestinal bacteria in the metabolism of salicylazosulfapyridine, The Journal of Pharmacology and Experimental Therapeutics, 181, 555 and 64, 240).
Another approach consists of exploiting bacterial hydrolases such as glycosidases and polysaccharidases (Friend D. R. (1995) Glycoside prodrugs: novel pharmacotherapy for colonic diseases, S.T.P. Pharma Sciences, 5, 70 Friend D. R. et al. (1984) A colon-specific drug-delivery system based on drug glycosides and the glycosidases of colonic bacteria, Journal of Medicinal Chemistry, 27, 261; Friend D. R. et al. (1985) Drug glycosides: potential prodrugs for colon-specific drug delivery, Journal of Medicinal Chemistry, 28, 51; and Friend D. R. et al. (1992) Drug glycosides in oral colon-specific drug delivery, Journal of Controlled Release, 19, 109). Prodrugs have thus been developed by coupling, for example, steroids to sugars (glucose, galactose, cellobiose, dextran (international patent application WO90/09168)), cyclodextrins (Hirayama F. et al. (1996) In vitro evaluation of Biphenylyl Acetic Acid-p-Cyclodextrin conjugates as colon-targeting prodrugs: drug release behavior in rat biological media, Journal of Pharmacy and Pharmacology, 48, 27).
3.2. Coating by Polymers Biodegradable by Bacterial Enzymes.
In this case, colonic targeting is done by coating the pharmaceutical form with a polymer specifically degraded by the enzymes produced by microflora, by benefiting from the presence of azoreductases or bacterial glycosidases.
Numerous polymers including azoaromatic links have been used to coat an active ingredient. Saffran et al. (Oral insulin in diabetic dogs, Journal of Endocrinology (1991), 131, 267 and A new approach to the oral administration of insulin and other peptide drugs, Science (1986), 233, 1081) have described the release of insulin and vasopressin in the colon of rats and dogs from oral forms coated with copolymers of styrene and hydroxyethylmethacrylate (HEMA) linked by azoaromatic bonds. This coating is degraded in the colon by bacterial azoreductases responsible for release of the active substance.
The advantage of azopolymers is that they allow very good colonic selectivity for release of active ingredients. The disadvantage associated with use is the lack of information on their possible toxicity.
To avoid this disadvantage, other studies have chosen to focus on the use of coating film based on natural substance such as polysaccharides in particular with coating films based on amylose/ethylcellulose (Milojevic S. et al. (1996) Amylose as a coating for drug delivery to the colon: preparation and in vitro evaluation using 5-aminosalicylic acid pellets, Journal of Controlled Release, 38, 75), based on dextrane ester (Bauer K. H. et al. (1995) Novel pharmaceutical excipients for colon targeting, S.T.P. Pharma Sciences, 5, 54) or pectin.
3.3. Matrices Biodegradable by Bacterial Enzymes.
Another approach of systems of specific release in the colon consists of the elaboration of matrices by compression of a mixture of active ingredient and biodegradable polymers such as chondroitin sulfate (Rubinstein A. et al. (1992b) Chondroitin sulfate: a potential biodegradable carrier for colon-specific drug delivery, International Journal of Pharmaceutics, 84, 141 and Rubinstein A. et al. (1992a) Colonic drug delivery: enhanced release of Indomethacin from cross-linked chondroitin matrix in rat cecal content, Pharmaceutical Research, 9,276), guar gum (Krishnaiah Y. S. R. et al. (1998) Evaluation of guar gum as a compression coat for drug targeting to colon, International Journal of Pharmaceutics, 171,137), chitosan (Tozaki H. et al. (1997) Chitosan capsules for colon-specific drug delivery: improvement of insulin absorption from the rat colon, Journal of Pharmaceutical Sciences, 86,1016) or pectin (Rubinstein A. et al. (1993) In vitro evaluation of calcium pectinate: a potential colon-specific drug delivery carrier, Pharmaceutical Research, 10, 258).
Systems based on enzymatic activity of microbial flora are probably those having the greatest colonic specificity for release of the active ingredients. Therefore they make up a future path for colonic targeting.
The interest in polysaccharides in the preparation of systems for colonic administration is that they are of natural origin, only slightly toxic and specifically degraded by bacterial enzymes of the colonic flora.
Thus, pectin is a polysaccharide isolated from the cellular walls of superior vegetables, widely used in the agro-alimentary industry (as a gelling agent or thickener of jams, ices . . . ) and pharmaceutical. It is polymolecular and polydisperse. Its composition varies according to the source, conditions of extraction and environmental factors.
Pectins are principally composed of linear chains of acids α-1,4-(D)-galacturonic, sometimes interspersed with units of rhamnose. The carboxylic groups of galacturonic acids can be partially esterified to give methylated pectins. Two types of pectin are distinguished according to their degree of methylation (DM: number of methoxy group per 100 units of galacturonic acid):                highly methylated pectin (HM: high methoxy) whereof the degree of methylation varies between 50 and 80%. It is only slightly soluble in water and form gels in an acid medium (pH<3.6) or in the presence of sugars;        slightly methylated pectin (LM: low methoxy), with a degree of methylafion from 25 to 50%. More soluble in water than HM pectin, it gives gels in the presence of divalent cations such as Ca2+ ions. In fact, Ca2+ ions form “bridges” between carboxylated groups free of galacturonic acids. The network thus formed has been described by Grant et al. Under the name of egg-box model (Grant G. T. et al. (1973) Biological interactions between polysaccharides and divalent cations: the egg-box model, FEBS Letters, 32, 195).        
There are also amidated pectins. Certain groups of methyl carboxylate (—COOCH3) can be transformed into carboxamide groups (—CONH2) by treatment of pectin by ammonia. This amidation imparts novel properties to the pectins, in particular improved resistance to variations in pH.
The pectin is degraded by enzymes originating from superior vegetables and diverse micro-organisms (mushrooms, bacteria . . . ) including bacteria of human colonic flora. The enzymes produced by the microflora are composed of polysaccharidases, glycosidases and esterases.
A galenic form is coated by pectin either via compression (Ashford M. et al. (1993b), An evaluation of pectin as a carrier for drug targeting to the colon, Journal of Controlled Release, 26, 213), or by pulverisation. Coating by compression is generally completed with pectin alone, whereas compression by pulverisation requires the use of a filmogenic polymer in addition to the pectin (Milojevic S. et al. (1996) Amylose as a coating for drug delivery to the colon: preparation and in vitro evaluation using 5-aminosalicylic acid pellets, Journal of Controlled Release, 38, 75; Wakerly Z. et al. (1996) Pectin/ethycellulose film coating formulations for colonic drug delivery, Pharmaceutical Research, 13, 1210).
Numerous matricial forms based on pectin have likewise been studied. They are generally constituted either by pure pectin, or by its complex with Ca2+ ions, slightly hydrosoluble, calcium pectinate. A matrix of calcium pectinate including indomethacin has in particular been described by Rubinstein et al. (1992a) Colonic drug delivery: enhanced release of Indomethacin from cross-linked chondroitin matrix in rat cecal content, Pharmaceutical Research, 9, 276) showing better stability of the calcium pectinate than the pectin alone in digestive juices, while remaining sensitive to the action of pectinolytic enzymes.
The amidated pectins, more tolerant to variations in pH have also been studied for elaboration of matricial tablets for colonic observation (Wakerly Z. et al. (1997) Studies on amidated pectins as potential carriers in colonic drug delivery, Journal of Pharmacy and Pharmacologyl. 49, 622).
Aydin et al.( (1996) Preparation and evaluation of pectin beads, International Journal of Pharmaceutics, 137,133) were the first to formulate pectin beads according to the ionic gelification method by Bodmeier et al. ((1989) Preparation and evaluation of drug-containing chitosan beads, Drug Development and Industrial Pharmacy, 15, 1475 and Spherical agglomerates of water-insoluble drugs, Journal of Pharmaceutical Sciences, 78, 964), who had disclosed beads of alginate and chitosan. Their objective was to incorporate in the beads two different active ingredients, a cationic (atenolol) and an anionic (piroxicam), so as to characterise possible interactions with pectin. They have thus demonstrated that it was possible to form beads with the 2 types of active ingredients and that the operational conditions had a major influence on the properties of the resulting beads.
Sriamornsak used beads of calcium pectinate to establish a system for specific release of proteins in the colon, by using bovine serum albumin (BSA) having a molecular weight of 66400 Da as protein model (Sriamornsak P. (1998) Investigation on pectin as a carrier for oral delivery of proteins using calcium pectinate gel beads, International Journal of Pharmaceutics, 169, 213 and (1999) Effect of calcium concentration, hardening agent and drying condition on release characteristics of oral proteins from calcium pectinate gel beads, European Journal of Pharmaceutical Sciences, 8, 221). He studied the influence of different factors of formulation on the properties of the resulting beads, such as their form, their size, the rate of encapsulation of the BSA and its release kinetics. Sriamornsak has therefore demonstrated that the pectinate beads of Ca could be employed for specific release of proteins in the colon. Obtaining an adequate release kinetic profile depends principally on the choice of the formulation and operational conditions for preparation of the beads. No in vitro/in vivo correlation of the release profiles of the encapsulated active ingredients has been established.
To boost the stability of the particles along the digestive tract and to avoid any premature release of the encapsulated active ingredient, it is possible to reinforce the pectin beads by reticulating them with a cationic polymer.
Munjeri et al.( (1997) Hydrogel beads based on amidated pectins for colon-specific drug delivery: the role of chitosan in modifying drug release, Journal of Controlled Release, 46, 273) have reticulated pectin beads amidated with chitosan. They then showed, by comparing the kinetics of dissolution of reticulated forms and of non-reticulated forms, that the chitosan allowed the release of the active insoluble ingredients to be minimised, but did not significantly modify the release of the hydrosoluble active ingredients. The loss of active ingredient in conditions emulating those of the stomach and the small intestine can therefore be limited by formation of a complex between the chitosan and the amidated pectin; the reticulated pectin beads still remain sensitive to the action of the colonic pectinolytic enzymes.
Another reticulating agent, polylysine, has been tested in the presence of alginate/pectin beads (Liu P. et al. (1999) Alginate/Pectin/Poly-L-lysine particulate as a potential controlled release formulation, J. Pharm. Pharmacol., 51,141). The beads reticulated by the polylysine seem to release less active ingredient in an acid medium (HCl O,1N) than the non-reticulated beads, except in the presence of highly hydrosoluble active ingredients. The same type of effect is found in an alkaline medium (phosphate buffer, pH 7.5) but it is clearly less marked than in acid medium.
International patent application WO 88/07865 suggests administering bacteria producing β-lactamases in the colon so as to hydrolyse the residual antibiotics. The micro-organisms utilised are bacteria with strict anaerobic metabolism, whereof the production and lyophilisation in sufficient quantity to make a drug are difficult. Furthermore, they are carriers of genes resistant to the antibiotics encoding for β-lactamases thus engendering a risk of dissemination of these genes within the colonic ecosystem and in the environment.
International patent application WO 93/13795 proposes an oral galenic form containing β-lactamases. It can be composed of saccharose particles of 1 to 2.5 mm in diameter enclosing the β-lactamases or amidase and optionally an inhibitor of trypsine, said particles being covered by a gastroresistant polymer. These particles could well release the enzyme in different segments of the digestive tract so that its activity takes place as required at the desired site in the intestine.
None of the examples comprises experimental data showing that the proposed galenic formulation is effectively capable of releasing the enzyme in an active form at the desired site in the intestine. In addition, no proof of the capacity of galenic preparation for effectively hydrolysing the antibiotic in vivo, nor even in vitro in a medium reproducing the characteristics of the intestinal medium is given.
For all these reasons, it is highly desirable to use a system for reducing the quantity of residual antibiotics which reach the colon after oral or parenteral antibiotherapy, or capable of delivering an active ingredient directly to the colon.
Therefore, the object of the present invention is multiparticular galenic forms to be utilised orally and for colonic delivery of active ingredients.